OPEN ACCESS Asian Journal of Biology

ISSN 1814-0068 DOI: 10.3923/ajcb.2016.1.12

Research Article Selective Cytotoxic Potential of IFN-γ and TNF-α on Breast Cell Lines (T47D and MCF7)

1Wahyu Widowati, 2Harry Murti, 1Diana Krisanti Jasaputra, 3Sutiman B. Sumitro, 4M. Aris Widodo, 5Nurul Fauziah, 5Maesaroh Maesaroh and 2Indra Bachtiar

1Medical Research Center, Faculty of Medicine, Maranatha Christian University, Jl. Prof. Drg. Suria Sumantri No. 65, Bandung, 40164, West Java, Indonesia 2Stem Cell and Cancer Institute, Jl A Yani No. 2 Pulo Mas, Jakarta, 13210, Indonesia 3Department of Biology, Faculty of Science, Brawijaya University, Malang, East Java, Indonesia 4Pharmacology Laboratory, Faculty of Medicine, Brawijaya University, Malang, East Java, Indonesia 5Biomolecular and Biomedical Research Center, Aretha Medika Utama, Bandung, Indonesia

Abstract Background: is one of the most common life threatening worldwide and it is the leading cause of death from cancer in women. The effectiveness of current cancer therapies is low, even though it requires expensive cost. Therefore, the development of the more efficient therapy is highly needed. Objective: This research was conducted to evaluate the effect Tumour Factor alpha (TNF-") and Interferon gamma (IFN-() as anticancer agents against breast cancer cells (T47D, MCF7) and selectivity of cytotoxic effect towards human Wharton’s Jelly Mesenchymal Stem Cells (hWJMSCs) to produce hWJMSCs-Conditioned Medium (CM), hWJMSCs-Cell Lysate (CL) and hWJMSCs which potential used as anticancer candidates after induced by TNF" or IFN(. Methodology: The hWJMSCs were isolated from umbilical cord and characterized by its surface marker phenotype and its multipotent differentiation potential. The breast cancer cell lines (T47D and MCF7) were treated by TNF-" and IFN-( and its cytotoxic activity was observed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)- 2H-tetrazolium (MTS) viability assay. The cytotoxic effects of TNF-" and IFN-( toward hWJMSCs were also observed using the same method. Results: Flow cytometry analysis showed that hWJMSCs for early passage (P4) were positive for hMSCs marker CD105, CD73 and negative for CD34, CD45, CD14, CD19 and HLA-II. It also showed the osteogenic, adipogenic and chondrogenic differentiation. The effect of TNF-" and IFN-( against MCF7 cells exhibited that the decreased the cell viability in a dose dependent manner. The IC50 value of TNF-" and IFN-( against breast cancer cell lines were found higher for T47D than MCF7. Conclusion: The TNF-" and IFN-( inhibit the cell growth of breast cancer cells due to apoptotic or necrotic cells but it non-toxic against hWJMSCs.

Key words: Breast cancer, TNF-α, IFN-γ, hMSCs, cytotoxic, umbilical cord

Received: August 26, 2015 Accepted: November 28, 2015 Published: December 15, 2015

Citation: Wahyu Widowati, Harry Murti, Diana Krisanti Jasaputra, Sutiman B. Sumitro, M. Aris Widodo, Nurul Fauziah, Maesaroh Maesaroh and Indra Bachtiar, 2016. Selective cytotoxic potential of IFN-γ and TNF-α on breast cancer cell lines (T47D and MCF7). Asian J. Cell Biol., 11: 1-12.

Corresponding Author: Wahyu Widowati, Medical Research Center, Faculty of Medicine, Maranatha Christian University, Jl. Prof Drg. Suria Sumantri No.65, Bandung, 40164, West Java, Indonesia

Copyright: © 2016 Wahyu Widowati et al. This is an open access article distributed under the terms of the creative commons attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.

Competing Interest: The authors have declared that no competing interest exists.

Data Availability: All relevant data are within the paper and its supporting information files. Asian J. Cell Biol., 11 (1): 1-12, 2016

INTRODUCTION cell free lysate (CL) of hWJMSCs demonstrate anticancer potential against solid tumour and become attractive Breast Cancer (BC) is the leading cause of death from candidates for future cancer therapies14,15. The CM of hWJMSCs cancer in women and one of the important contributors to the (hWJMSCs-CM) incubated in normoxic and hypoxic conditions global health burden1. It is the second leading cause of death can inhibit proliferation of various cancer cell lines, including by cancer, which is 1 in 3 diagnosed among women cervical (HeLa), liver (HepG2), prostate (PC-3), ovarian (SKOV3) in the United States2 and also the second most frequent and oral squamous (HSC3) cancer cell lines and were safe for among women in Mexico. In 2008, breast cancer was the most normal cells in human fibroblast, mouse (NIH3T3) and human incident of cancer and a major cause of mortality from cancer Mesenchymal Stem Cells (hMSCs)15. The hMSCs are the among women in ASEAN3. Cancer has a severe impact on attractive vehicles to deliver therapeutic agents in various individuals and community which leads to disability and tumour sites to introduce expression or to secrete the death. Its high treatment costs, which are associated with therapeutic factor for cancer and to enhance the anticancer higher income loss can quickly undermine family finances3. activity16. Anticancer agents produced by engineered The development of antineoplastic drugs have been based MSCs are classified into: (a) Immunostimulation including on the conviction that cancer would constitute a chemokine C-X3-C motif ligand 1 (CX3CL1), interferon (IFN) cell-autonomous genetic and epigenetic disease4. Even and interleukins (IL2, IL7 and IL12), (b) Pro-drug conversion, though there are improved treatment models, many tumours such as cluster of differentiation (CD) and Herpes Simplex remain unresponsive to the current conventional cancer Virus Thymidine Kinase (HSV-tk) and (c) induction therapies including surgery, chemotherapy and radiotherapy5. such as IL-8, Natural Killer 4 (NK4) and tumour necrosis The effectiveness of current cancer therapies is low although factor-related apoptosis inducing ligand (TRAIL)5,10,17. it requires high cost6. Therefore, the development of the more The TNF-" as an anticancer drug is limited to local efficient therapy is highly needed. Strategic therapy can target treatments because of its dose-limiting systemic toxicity18. The tumours directly, both in primary and metastatic side7. TNF-" can induce apoptosis but also supports cell survival Tissue engineering has also become the new promising pathways19. Although TNF-" is cytotoxic to certain tumour cell therapies. Human Mesenchymal Stem Cells (hMSCs) are the lines, it does not trigger apoptosis in normal cells but instead sources of adult stem cells for cell therapy and tissue stimulates the proliferation of normal fibroblasts20. The type II engineering8. The hMSCs are the powerful source for tissue Interferon (IFN-() is an essential cytokine for repair, because it has the multi-potency differentiation immunity against intracellular pathogens and in controlling capability, easy to acquire, easy harvesting process and tumour cell growth21 but IFN-( may also facilitate culture, fast expansion, the feasibility of autologous progression22. Continuing research was conducted to evaluate and allogenic therapy and a powerful paracrine function9. The the effect TNF-" and IFN-( as anticancer agents against breast hMSCs are capable to migrate, incorporate into the tumour cancer cells (T47D, MCF7) and selectivity of cytotoxic effect site both in vitro co-culture and in vivo xenograft and survive towards hWJMSCs to produce CM (hWJMSCs-CM) and CL in cancer tissue5,7,8. The hMSCs can be delivered successfully (hWJMSCs-CL) and hWJMSCs as anticancer candidate induced for various treatments to treat multiple metastatic cancers, by TNF " or IFN(. including lung, breast, squamous cell, colon, pancreas and cervical cancers10,11. The hMSCs can be isolated from adult MATERIALS AND METHODS tissues, including bone marrow, peripheral blood, adipose tissue, skeletal muscle, synovium, dental pulp and neonatal Isolation and cell culture of hWJMSCs: Isolation and birth-associated tissues including umbilical cord blood, cultivation of hWJMSCs from umbilical cord was as umbilical cord, placenta, decidua, chorion villi, chorion described by Widowati et al.15,23. Fresh human Umbilical Cords membrane, amniotic membrane, amniotic fluid and Wharton’s (UC, n = 3) from women aged 25-40 years after full-term births Jelly (WJ)12. Wharton’s Jelly (WJ) is part of the umbilical cord, (normal vaginal delivery), all donors signed an informed as one source of hMSCs has many advantages including low consent and research guidelines using UC were approved by risk of infection, non-carcinogenesis, multipotency and low Ethics Committee at the Stem Cell and Cancer Institute, immunogenicity13. The hWJMSCs have anticancer potential Jakarta, Indonesia and from Ethics Committee collaboration which mediated via cell-to-cell and/or non-cellular contact between Maranatha Christian University, Bandung, Indonesia mechanism. The hWJMSCs, Conditioned Medium (CM) and and Immanuel Hospital Bandung, Bandung, Indonesia.

2 Asian J. Cell Biol., 11 (1): 1-12, 2016

The hWJMSCs from UC were rinsed in normal saline (Gibco A10071-01, Invitrogen) for 2 weeks. Chondrocytes were (0.9% w/v sodium chloride) and cut into very small explants visualized using Alcian blue (Amresco, 0298). Adipogenic (1-2 mm), then plated on tissue culture plastic plates. The differentiation of hWJMSCs was done using StemPro explants were cultured in Minimum Essential Medium-" Adipogenesis Differentiation Kit (Gibco A10070-01, Invitrogen, (MEM-") with 2 mM GlutaMAX (Invitrogen, Carlsbad, CA, USA), Carlsbad, CA, USA) for 2 weeks. We used Oil Red O supplemented with 20% Fetal Bovine Serum (FBS, Invitrogen) (Sigma 00625, St Louis, USA) to confirm the lipid droplets23. and penicillin, streptomycin amphotericin B (100 U mLG1, 100 and 0.25 mg mLG1; Invitrogen). Cell cultures were Cell viability assay of cancer cells-treated TNF-" and IFN-(: ® incubated in a humidified atmosphere with 5% CO2 at 37EC Breast cancer cell lines MCF7 (ATCC HTB22™) and T47D and medium was replaced every 5 days for 21 days. The cells (ATCC®HB133™) were obtained from Stem Cell and Cancer were harvested and replated at a density 8×103 cells cmG2, Institute, Jakarta Indonesia. Medium consist of Roswell Park when cells reached 80-90% confluence. The hWJMSCs were Memorial Institute (RPMI) (Biowest L0495) for T47D and 15,23 cultured in 95% relative humidity (21% O2) and 5% CO2 . Dulbecco’s Modified Eagle’s Medium (DMEM) (Biowest L0104) for MCF7 supplemented with FBS 10% (Biowest S1810) and Cell surface phenotype and multipotent differentiation: 1% penicillin-streptomycin (Biowest L0018), then incubated in 3 The surface marker characterization, including CD105, CD73, 5% CO2, 37EC incubator. Cells were seeded at density of 5×10 CD90, CD34, CD45, CD14, CD19 and Human Leukocyte in 96 well plate for 24 h incubation after cells reached 80% II (HLA-II) was performed to confirm the phenotype confluence. Cells were supplemented with TNF-" characteristic of hWJMSCs of passage (P4) using a flow recombinant (Biolegend 570106) and IFN-( recombinant cytometry. The hWJMSCs at 80% confluence were harvested (Biolegend 570206) in various concentrations (0, 60, 180, 260 and dissociated with trypsin-EDTA, then centrifuged at 300 g and 520 ng mLG1) then incubated for 24, 40 and 72 h. The MTS for 10 min. The pellet was resuspended with Phosphate (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxyme-thoxyphenyl)-2- Buffered Saline (PBS)+2% FBS and cells were counted with a (4-sulfophenyl)-2H-tetrazolium) (Promega, Madison, WI, USA) hemocytometer. Around 100-200 cells in 25 mL PBS were was added at 10 µL to each well. The plate was incubated at stained with the appropriate surface monoclonal . 5% CO2, 37EC for 4 h. The absorbance of the cells was Isotype controls were used to determine background staining. measured at 490 nm using a micro-plate ELISA reader These antibodies were as follows: PE (Phycoerythrin) (MultiSkan Go). The data were presented as the number of conjugated: CD105 (abcam 53321-100), CD73 (BD550257), viable cells, the percentage of viable cells (%), the inhibition of CD90 (abcam 226), CD34 (BD 348053), CD45 (BD 555482), cell proliferation (%) and the median Inhibitory Concentration 15,24,25 CD14 (abcam 28061-100), CD19 (abcam 1168-500) and (IC50) were calculated from the data . HLA-DR (abcam 23901), FITC conjugated: mIgG1 (BD349041 and BD 349043) and mIgG2 (BD349053) then Cell viability assay of hWJMSCs-treated TNF-" and IFN-(: added to each FACS tube: isotype mIgG2a-PE, CD105-PE, Cells viability of hWJMSCs was determined by using MTS HLA class II-PE, isotype mIgG1-PE, CD73-PE, CD19-PE; isotype (Promega, Madison, WI, USA). Cells were maintained at mIgG1-FITC, CD34-FITC, CD45-FITC, CD14-FITC after MEM-" (Biowest L0475) with 2 mM GlutaMAX incubation at 4EC for 15 min. The cells were analyzed by flow (Invitrogen, Carlsbad, CA, USA), supplemented with 20% FBS cytometry with a FACS Calibur TM 3 argon laser 488 nm (Biowest S1810) and penicillin streptomycin-amphotericin B (Becton Dickinsok Biosciences, Franklin Lakes, NJ, USA) using (100 U mLG1, 100 and 0.25 mg mLG1; Invitrogen). Cell cultures CellQuest Pro Acquisition on the BD FACS tation TM Software. were incubated in an incubator (humidified atmosphere, 3 The experiments and measurement of surface marker were 5% CO2 at 37EC). Cells were seeded at density of 5×10 in performed in triplicate15,23. 96 well plate for 24 h incubation after cells reached 80% For osteogenic differentiation, hWJMSCs (P4) were confluence. Cells were supplemented with TNF-" seeded at density 1×104 cells cmG2 in culture dishes using recombinant (Biolegend 570106) and IFN-( recombinant StemPro Osteogenesis Differentiation Kit (Gibco A10072-01, (Biolegend 570106) in various concentrations (0, 60, 180, 260, Invitrogen) for 3 weeks. Calcium deposits were visualized 520 ng mLG1) then incubated for 72 h. The MTS was added at using Alizarin red S (Biochemicals and Life Science Research 10 µL to each well and incubated at 5% CO2, 37EC for 4 h. The Products, Amresco 9436). For chondrogenic differentiation, absorbance of the cells was measured at 490 nm using a hWJMSCs were seeded at density 1×104 cells cmG2 in culture micro-plate ELISA reader (MultiSkan Go). The data were dishes using Stem Pro Chondrogenesis Differentiation Kit presented as number of viable cells, the percentage of viable

3 Asian J. Cell Biol., 11 (1): 1-12, 2016 cells (%), the percentage of proliferative inhibition (%) and the Research Products, Amresco 9436). The hWJMSCs were median Inhibitory Concentration (IC50) were calculated from cultured in chondrogenesis differentiation medium the data15,24,25. (StemPro Chondrogenesis Differentiation Kit, Gibco A10071-01, Invitrogen) for 2 weeks and the differentiated Statistical analysis: The data of cell number, cell viability and hWJMSCs exhibited chondrocyte expression using Alcian blue proliferative inhibition of hWJMSCs, MCF7 and T47D cells were (Amresco, 0298). After 2 weeks, hWJMSCs were cultured in calculated and expressed in means and standard deviation adipogenesis differentiation (StemPro Adipogenesis (M±SD). Analysis of variance (ANOVA) was used to compare Differentiation Kit, Gibco A10070-01, Invitrogen), the among treatments with p<0.05 as statistically significant and differentiated cells exhibited lipid droplets was observed by continued with Tukey honestly significant difference post hoc using Oil Red O (Sigma 00625, St Louis, USA) (Fig. 2). Cell test and 95% confidence interval. The median Inhibitory staining showed that hWJMSCs from three donors were able

Concentration (IC50) was analyzed by using probit analysis to to differentiate into chondrocytes, osteocytes and adipocytes determine the cytotoxic effect of TNF-" and IFN-( on cells (Fig. 2)15. (hWJMSCs, MCF7 and T47D cells). Statistical analysis was performed in SPSS version 20.0 program (IBM SPSS Statistics Cytotoxic effect of TNF-" and IFN-( in breast cancer cells: for Windows, Version 20.0. Armonk, NY: IBM Corp, SPSS Inc., The viability assay was performed to evaluate the effect of 15 Chicago, IL, USA) . TNF-" and IFN-( against breast cancer cells (MCF7 and T47D). The breast cancer cells were cultured in a 96-well plate with RESULTS density 5×103 cells cmG2. The cytotoxic effect of TNF-", IFN-( were determined by the cells number, the cell viability MSC markers by cell surface phenotype: Flow cytometry according to the MTS assay. Human recombinant TNF-" and analysis showed that hWJMSCs for P4 were positive for hMSCs IFN-( demonstrated reducing cancer cell proliferation in a marker CD105, CD73 and negative for CD34, CD45, CD14, dose dependent manner. The effect of TNF-" and IFN-( CD19 and HLA-II. The surface marker of hWJMSCs can be seen against T47D cells in cells number, cells viability and in Table 1 and Fig. 1. The surface marker of hWJMSCs from proliferation inhibition can be seen in Table 2. The number of three donors show the characteristic of hWJMSCs by high T47D cells increased at low concentration but at higher expression of CD105, CD73 markers (>94%) and low concentration of TNF-" and IFN-( killed and inhibited the cells expression of CD34, CD45, CD14, CD19 and HLA-II markers proliferation. The effect of TNF-" and IFN-( against MCF7 cells (<2%)15,23. in cells number, cells viability and proliferative inhibition can be seen in Table 3. The effect of TNF-" and IFN-( against MCF7 Multilineage differentiation capacity of hWJ-MSCs: The cells exhibited that the cytokines decreased the cell multilineage differentiation of hWJMSCs cultured in MEM-" was evaluated by culturing hWJMSCs in the differentiation Table 1: Surface marker of hWJMSCs for passage 4 medium (chondrogenic, osteogenic and adipogenic Surface marker Donor 1 Donor 2 Donor 3 CD73 96.62±2.12 97.62±0.96 98.35±0.29 lineages).The hWJMSCs were cultured in osteogenesis CD105 93.73±0.36 95.14±1.35 95.13±2.50 differentiation medium (StemPro Osteogenesis Differentiation HLA-DR -3.68±0.45 -2.90±2.01 -3.64±1.88 Kit, Gibco A10072-01, Invitrogen) for approximately 3 weeks CD14 0.01±0.01 0.00±0.01 0.00±0.01 with the osteocytes morphology were assayed. The CD19 -1.54±1.38 -0.81±0.75 -0.61±0.07 CD34 0.00±0.01 0.00±0.01 0.00±0.00 differentiated cells exhibited calcium deposits based on the CD45 0.00±0.01 0.00±0.01 0.00±0.01 staining of Alizarin red S (Biochemicals and Life Science Data was presented as Mean±SD of surface markers of hWJMSCs

Table 2: Effect of TNF-" and IFN-( against breast cancer cells (T47D) for 72 h incubation TNF-" IFN-( ------Concentrations (ng mLG1) No. of cells Cells viability (%) Cells inhibition (%) No. of cells Cells viability (%) Cells inhibition (%) 0 3230±443b 100.00±0.00c 0.00±0.00a 3230±443d 100.00±0.00bc 0.00±0.00ab 60 3318±331b 103.14±4.78c -3.14±4.78a 3987±175e 124.92±17.08c -24.92±17.08a 180 2484±448b 77.13±10.48b 22.87±10.48b 2491±85c 78.21±12.37b 21.79±12.37b 260 871±46a 27.38±4.79a 72.62±4.79c 1395±143b 43.82±8.25a 56.18±8.25c 520 633±125a 20.09±6.09a 79.91±6.09c 553±146a 16.91±2.44a 83.09±2.44c Data are expressed as Mean±SD, different superscripts of small a, ab, b, bc, c, d, eletters in the same column (among concentrations of TNF-" and IFN-( treatment on T47D cells) were significantly different at p<0.05 (Tukey honestly significant difference post hoc test), TNF-": Tumour necrosis factor alpha, IFN-(: Interferon gamma

4 Asian J. Cell Biol., 11 (1): 1-12, 2016

200 200 (a) (b)

160 160

120 120 Counts Counts 80 80

40 40

0 0 0 1 2 3 4 100 101 102 103 104 10 10 10 10 10 FL2-H FL2-H 200 (c) 200 (d)

160 160

120 120

Counts 80 80 Counts

40 40

0 0 100 101 102 103 104 100 101 102 103 104 FL1-H FL1-H 200 (e) 200 (f)

160 160

120 120 Counts

Counts 80 80

40 40

0 0 100 101 102 103 104 100 101 102 103 104 FL2-H FL2-H

200 (g)

160

120 Counts 80

40

0 100 101 102 103 104 FL1-H

Fig. 1(a-g): Representation of flow cytometry measurement on (a) CD105, (b) HLA-II, (c) CD34, (d) CD45, (e) CD73, (f) CD19 and (g) CD14, hWJMASCs: Human Wharton’s jelly mesenchymal stem cells

Table 3: Effect of TNF-" and IFN-( against breast cancer cells (MCF7) for 72 h incubation TNF-" IFN-( ------Concentrations (ng mLG1) No. of cells Cells viability (%) Cells inhibition (%) No. of cells Cells viability (%) Cells inhibition (%) 0 15380±1.168d 100.00±0.00d 0.00±0.00a 15380±1.168c 100.00±0.00c 0.00±0.00a 60 11951±96c 79.53±4.84c 20.47±4.84b 9050±1.643b 58.18±6.80b 41.82±6.80b 180 9550±213b 63.78±2.27b 36.22±2.27c 7221±129ab 48.09±2.57a 51.91±2.57bc 260 7691±892a 52.46±8.56ab 47.44±8.56cd 7135±801ab 45.88±3.94a 54.12±3.94c 520 6900±375a 45.35±2.86a 54.65±2.86d 5832±333a 38.58±3.40a 61.42±3.40c Data are expressed as Mean±SD, different superscripts of small a, ab, b, bc, c, cd, dletters in the same column (among concentrations of TNF-" and IFN-( treatment on MCF7 cells were significantly different at p<0.05 (Tukey honestly significant difference post hoc test), TNF-": Tumour necrosis factor alpha, IFN-(: Interferon gamma

5 Asian J. Cell Biol., 11 (1): 1-12, 2016

(a) (b)

(c)

Fig. 2(a-c): Morphological appearance of (a) Osteogenic (b) Adipogenic and (c) Chondrogenic differentiation of hWJMSCs from three donors at P4, hWJMSCs: Human Wharton’s jelly mesenchymal stem cells

Table 4: IC50 of TNF-" and IFN-( against breast cancer cells (T47D and MCF7) hWJMSCs were carried out in various concentrations of T47D IC (ng mL 1)IC (µg mL 1) 50 G 50 G cytokines (0, 60, 100, 180, 260 and 520 ng mLG1), which TNF-" 242.77 0.24 hWJMSCs were treated with cytokines for 1, 2, 3 days. The IFN-( 266.88 0.27 1 1 selective cytotoxic effect of TNF- , IFN- against hWJMSCs MCF7 IC50 (ng mLG )IC50 (µg mLG ) " ( TNF-" 364.78 0.36 were determined, including the cells number, the cell viability IFN-( 295.03 0.30 by MTS assay, it can be seen in Table 5 and 6. The IC50 value of IC is the median inhibitory concentration of TNF-" and IFN-( against the T47D 50 TNF-" and IFN-( against hWJMSCs after 72 h incubation were and MCF7, TNF-": Tumour necrosis factor alpha, IFN-(: Interferon gamma found infinite (Table 7). Based on the Table 7 both TNF-" and IFN-( are non-toxic toward hWJMSCs. viability in a dose dependent manner. The IC50 value of TNF-" and IFN-( against breast cancer cell lines were found DISCUSSION 242.77-266.88 ng mLG1 for T47D and 295.03-364.78 ng mLG1 for MCF7 can be seen in Table 4. Based on the Table 4 both The surface marker of hWJMSCs from 3 donors showed TNF-" and IFN-( are more active as anticancer in T47D cells positive results for CD105, CD73 and negative for CD34, CD45, compared to MCF7. CD14, CD19 and HLA-II (Table 1). These data were validated by the previous result that hWJMSCs highly expressed CD105, Cytotoxic effect of TNF-" and IFN-( in mesenchymal stem CD73 and low expression of CD34, CD45, CD14, CD19 and cells: The cells viability assay of hWJMSCs after TNF- and " HLA II15,22. The hWJMSCs were able to differentiate into IFN-( treatment were performed to determine the selective chondrocyte, osteocyte and adipocyte (Fig. 1) as previous cytotoxic effect of TNF-" and IFN-( against only to cancer cells research15. The TNF-" and IFN-( inhibited the growth rate of and non-toxic against to normal cells. The TNF-" and IFN-( are breast cancer cells both in T47D and MCF7 in a dose non-toxic, safe and can induce or increase the anticancer dependent manner, higher concentration of cytokines would potency of mesenchymal stem cells. This research was carried decrease number of cells, cell viability and increase out to evaluate the cytokines TNF-" and IFN-( as an anticancer proliferation inhibition (Table 3). The TNF-" and IFN-( drug directly toward cancer cells but these cytokines are safe inhibited the breast cancer cells induced by apoptosis or cell against hWJMSCs. The effect of TNF-" and IFN-( against death.

6 Asian J. Cell Biol., 11 (1): 1-12, 2016

Table 5: Effect of TNF-" against hWJMSCs in various incubations and concentrations Concentrations (ng mLG1) ------Incubation 0 60 100 180 260 520 Number of cells hWJMSCs-TNF-" Day 1 9422±135aA 11217±48bA 9133±496aA 9268±536aA 9388±589aA 9320±411aA Day 2 15435±456bcC 15425±264bcC 15503±588cC 14054±188abB 14071±51abcB 13227±995aB Day 3 11948±699bB 12898±3.11cB 10919±199abB 9648±188aA 9958±91aA 9943±49aA Viability of hWJMSCs-TNF-" (%) Day 1 100.00±0.00aA 119.08±2.20bA 96.96±5.68aA 98.43±6.96aA 99.61±5.08bA 98.89±3.12aA Day 2 163.89±6.93cdC 163.74±3.21bcdC 164.51±3.97dC 149.17±0.84abB 149.37±2.10abcB 140.35±9.52aB Day 3 126.77±5.97cB 136.89±2.54dB 115.93±3.48bB 102.42±1.36aA 105.70±1.43aA 105.54±1.00aA Inhibition of hWJMSCs-TNF-" (%) Day 1 0.00±0.00bC -19.08±2.20aC 3.04±5.68bC 1.57±6.96bA 0.39±5.08bB 1.11±3.12bB Day 2 -63.89±6.93abA -63.74±3.21abcA -64.51±3.97aA -49.17±0.84cdB -49.37±2.10cdA -40.35±9.52dA Day 3 -26.77±5.97bB -36.89±2.53aB -15.93±3.48cB -2.42±1.36dA -5.70±1.43dB -5.54±1.00dB Data are presented as Mean±SD, a, ab, abc, b, bc, c, cd, dDifferent superscript of small letter are significant differences among the means of groups (among concentrations of TNF-" treatment) in same row, A,B,CSignificant differences among the means of groups (among long incubation day 1, 2 and 3) in number of cells, cells viability, cells proliferation inhibition in same column at p<0.05 based on Tukey honestly significant difference post hoc test, TNF-": Tumour necrosis factor alpha, hWJMSCs: Human wharton’s Jelly mesenchymal stem cells

Table 6: Effect of IFN-( against hWJMSCs in various incubations and concentrations Concentrations (ng mLG1) ------Incubation 0 60 100 180 260 520 Number of cells hWJMSCs-IFN-( Day 1 10210±607abA 12817±1.960bA 9814±687aA 9851±621aA 9793±953aA 9744±627aA Day 2 10922±1.122aA 19175±629cB 16246±205bB 14890±984bC 10218±547aA 9749±15aA Day 3 10093±278aA 15340±1.060cA 12198±422bB 12046±523bB 11232±257abA 10005±344aA Viability of hWJMSCs-IFN-( Day 1 100.00±0.00aA 126.370±25.09aA 96.130±3.72aA 96.560±4.96aA 96.170±11.07aA 95.420±1.02aA Day 2 107.68±17.78aA 188.160±10.55cB 159.460±8.79bcC 146.120±11.48bB 100.540±11.72aA 95.710±5.75aA Day 3 98.98±3.44aA 150.740±15.73bAB 119.710±7.16aB 118.330±10.05aA 110.180±4.45aA 98.190±45.78aA Inhibition of hWJMSCs-IFN-( Day 1 0.00±0.00aA -26.370±25.09aB 3.870±3.72aC 3.440±4.96aB 3.830±11.07aA 4.580±1.02aA Day 2 -7.68±17.78cA -88.160±10.55aA -59.460±8.79abA -46.120±11.48bA -0.540±11.72cA 4.290±5.75cA Day 3 1.02±3.44bA -50.740±15.73aAB -19.710±7.16bB -18.330±10.05bB -11.300±1.81bA 0.820±4.16bA Data are presented as Mean±SD, a, ab, b, bc, cDifferent supercript of small letters are significant differences among the means of groups (among concentrations of TNF-" treatment) in same row, A,B,CSignificant differences among the means of groups (among long incubation day 1, day 2, day 3) in number of cells, cells viability, cells proliferation inhibition in same column at p<0.05 based on Tukey honestly significant difference post hoc test, TNF-": Tumour necrosis factor alpha, hWJMSCs: Human Wharton’s Jelly mesenchymal stem cells

29,30 Table 7: IC50 of TNF-", IFN-( against hWJMSCs internalization and endosomal trafficking . Complex II leads Incubation TNF-" IFN-( to the death-inducing signalling complex formation and Day 3 (72 h) ~ ~ ultimately, -dependent apoptosis31. ~: Infinite, TNF-": Tumour necrosis factor alpha, IFN-(: Interferon gamma, hWJMSCs: Human Wharton’s jelly mesenchymal stem cells, IC: Inhibitory The direct targeting potential of TNF-" in tumour cells has concentration been clinically used recently in therapy designs such as for soft tissue sarcoma and with an acceptable safety Tumour Necrosis Factor (TNF) super family is a group of profile32,33. The TNF-" was reported as anticancer therapy due cytokines that have many important functions in process of to its cytotoxic effect against a number of tumour cells32,34,35. immunity, inflammation, cell differentiation, cell survival, cell Blocking the intracellular cell survival pathways that activated proliferation and apoptosis26,27. The binding of TNF-" to its by TNF-" definitely leads tumour cells to apoptosis33,36. The receptor, which is Tumour Necrosis Factor Receptor (TNFR1) apoptotic process is complex and mainly triggered through can alternatively induce cell death or survival and the activation of death receptors37, among these the Tumour differentiation through the formation of two sequential Necrosis Factor Receptor1 (TNFR1) and its ligand TNF-" play complexes28. Complex I triggers rapid activation of the a crucial role38. The binding of TNF-" to TNFR1 triggers a series factors Nuclear Factor-kappa B (NF-kB) and of intracellular events initiated by the recruitment of a key activator -1, complex II is formed after the former adaptor protein TNFR1-associated with death domain protein is released from the membrane into the cytosol by receptor (TRADD) to the receptor complex26,39.

7 Asian J. Cell Biol., 11 (1): 1-12, 2016

The TNF-" induces caspase-dependent (apoptotic) and showed cytostatic and cytotoxic effects on cell lines previously caspase-independent (necrosis-like) cell death in different resistant to TNF-" and IFN-( individually34. The IFN-( has an types of cells40. play key roles in mediating Fas or important role in protective mechanisms against viral, TNF-"-induced apoptosis and are divided into two classes bacterial infections and tumour control55,56, antiviral, anti- based on the lengths of their N-terminal prodomains: tumour and immunomodulatory activities57. The IFNs mediate upstream caspases such as caspase 8 and 10 and downstream anti-tumourigenic effects indirectly by modulating caspases41 such as -3, -6 and -7. The activation of caspases are immunomodulatory responses or directly by regulating required for apoptosis41. The FAS Associated Death Domain tumour cell proliferation and differentiation58 and inhibition of Protein (FADD) is essential for TNF-induced apoptosis as it tumour angiogenesis59. recruits caspase-842,43. Caspase-8 is activated, thereby initiating The IFN-( treatment induced phosphorylation of ATR a caspase cascade, which results in apoptosis26,44-46. The BID is (ataxia-telangectasia and Rad3-related protein) and Chk1 a target of caspase-847,48. The TNF-" is able to induce apoptosis (Serine/-specific protein kinase that in humans), via two distinct caspase-8 activation pathways, which are which activated the ataxia-telangectasia by ATR and Chk1 differentially regulated by Cellular inhibitor of apoptosis signalling following the treatment. The ATR-Chk1 signalling 1 and 2 (cIAP1/2) and Cellular FLICE-like inhibitory might be associated with IFN-(-induced responses. protein (c-FLIP)49. The TNF-induced necrotic cell death Phosphorylation of both ATR and Chk1 increased in the through NF-kB activation are the anti-apoptotic RIP-, TRAF2- presence of IFN-( in a dose-dependent manner57. and FADD-mediated (ROS) Dose-dependent decrease of Chk1 protein was also observed accumulation46. Reactive Oxygen Species was found to be following the IFN-( treatment. The IFN-( treatment decreased required for necrotic cell death in L929 cells47. The TNF-" can the half-life of Chk1 indicating that Chk1 protein was also induce cell death in a context-dependent manner50. The destabilized following IFN-( treatment57. The direct effect of TNF-induced cell death involves the BCL2/Adenovirus E1B 19 IFN-( on tumour cell killing is inhibiting cellular proliferation kDa interacting protein 3 (BNIP3), ROS production and mediated by Signal Transducers and Activators of activation of the lysosomal death pathway40. Apoptosis Transcription 1 (STAT-1), Interferon Regulatory Factor-1 (IRF-1), induced by TNF-" is also associated with the generation of p21 or p2760-62. The IFN-( has anti-proliferative effects on ROS. Induction of ROS production or an inhibition of the NF-kB cancer cells. The numerous anti-proliferative effects of IFN-( in pathway by cyclooxygenase-2 (COX-2) inhibitors is reported many cancers through G1 arrest involving down-regulation of to be successful in sensitizing tumour cells to TNF-"-induced G1/S cyclins (cyclins A and E)63 and CDK2/4. Treatment using apoptosis51. IFN-( induces the up regulation of pro-apoptotic proteins such Based on Table 2 and 3, TNF-" could induce proliferation as Fas, Fasligand (FasL) and TRAIL58,64,65. These proteins can and inhibition at 72 h of incubation period. This result was interact with FADD or TRAIL-receptor proteins, resulting in validated with previous research that TNF-" showed a initiation of apoptosis through activation of caspase-866,67. The dose- and time-dependent cytotoxic effect on cell survival. IFN-induced apoptosis involves the FADD/caspase-8 signalling The longer the periods of time, the cell mortality increased pathway, activation of the caspase cascade, release of even further. The MCF7 stimulated TNF-" increased the mitochondrial cytochrome c, disruption of mitochondrial phosphorylated state of Akt (pAkt)52. Treatment using membrane potential, changes in plasma membrane symmetry 20 ng mLG1 of TNF-" in HeLa and HepG2 cell lines for 24 h and DNA fragmentation 58,68. The IFN-( exposure also increased incubation could not inhibit the cell proliferation or no dead caspase-8 expression in three of six cell lines69. Many of the cell observed32. Low dose of TNF-" induced the cell survival inhibitory effects of IFN( appeared to be mediated by IRF-1. while the high dose of TNF-" (100 ng mLG1) inhibited the cell The IRF-1 also regulates the expression of several survival (10%)49. This previous research showed consistent involved in apoptosis, such as the pro-apoptotic protein BAX, with this data, Table 2 shows that low concentration of TNF-" the tumour suppressor , caspase-1 and interferon-induced (60 ng mLG1) could induce cell proliferation in T47D cells. protein kinase (PKR)70. The anti-proliferative action of IFN-( The TNF-" also demonstrated to enhance the anti-tumour depends in part on STAT1, which induces the expression of effect in vitro, when used in combination with other the cell cycle inhibitor, cyclin dependent kinase inhibitor 1A cytokines34,53. When combined with interferon alpha and (CDKN1A) (p21CIP1), preventing entry into the of the gamma (IFN-" and IFN-(), TNF-" induced synergistic growth cell cycle60. The IFN(-induced growth suppression can be inhibition toward pancreatic cancer cell lines54. The rescued by blocking Ras GTPase71 and reducing p53 or the combination of TNF-" and IFN-( on 23 cell lines in vitro, Ataxia-Telangectasia Mutated (ATM) kinase protein levels57,72.

8 Asian J. Cell Biol., 11 (1): 1-12, 2016

The stimulation of TNF-" and IFN-( did not inhibit the REFERENCES hWJMSCs proliferation. This data were consistent with previous research that MSCs-prestimulated IFN-( and TNF-" 1. Soerjomataram, I., J. Lortet-Tieulent, D.M. Parkin, J. Ferlay, significantly reduced expression of Transforming Growth C. Mathers, D. Forman and F. Bray, 2012. Global burden of cancer in 2008: A systematic analysis of disability-adjusted Factor beta (TGF-$)73, a secreted protein that controls cell life-years in 12 world regions. Lancet., 380: 1840-1850. proliferation, cellular differentiation and other functions in 2. DeSantis, C., R. Siegel, P. Bandi and A. Jemal, 2011. Breast most cells. The TGF- acts as an anti-proliferative factor in $ cancer statistics, 2011. CA: A Cancer J. Clin., 61: 408-418. 74 normal epithelial cells and at early stages of oncogenesis . 3. Kimman, M., R. Norman, S. Jan, D. Kingston and The IFN-( stimulation (500 U mLG1) in hMSCs elevated the M. Woodward, 2012. The burden of cancer in member Indolamine 2,3-dioxygenase (IDO) expression and increased countries of the Association of Southeast Asian Nations the iNOS levels in mouse MSCs lysate75. The IDO in inhibiting (ASEAN). Asian Pac. J. Cancer Prev., 13: 411-420. alloantigen drives proliferation by stimulated MSCs73. 4. Hanahan, D. and R.A. Weinberg, 2011. Hallmarks of cancer: The next generation. Cell, 144: 646-674. The IFN-( prestimulated MSCs (IMSCs) maintained 5. Sun, X.Y., J. Nong, K. Qin, G.L. Warnock and L.J. Dai, 2011. their in vitro differentiation capacity, forming osteoblasts, Mesenchymal stem cell-mediated cancer therapy: 75 adipocytes and chondroblasts . Human MSCs cultured for 6 A dual-targeted strategy of personalized medicine. days in the presence of IFN-( retained their capacity to adhere World J. Stem Cells, 3: 96-103. to plastic75. The pre-treatment of MSCs prior to transplantation 6. Siddiqui, M. and S.V. Rajkumar, 2012. The high cost of cancer with TNF-" increased adhesiveness and migration of MSCs drugs and what we can do about it. Mayo Clin. Proc., in vitro, thus lead to increased expression of Bone 87: 935-943. Morphogenetic Protein (BMP)-2 by MSCs72. 7. Yang, Z.S., X.J. Tang, X.R. Guo, D.D. Zou and X.Y. Sun et al., 2014. Cancer cell-oriented migration of mesenchymal stem cells engineered with an anticancer gene (PTEN): An imaging CONCLUSION demonstration. Onco. Targets Ther., 17: 441-446. 8. Bruder, S.P., K.H. Kraus, N. Grafton, V.M. Goldberg and This preliminary research about the effect of TNF-" and S. Kadiyala, 1998. The effect of implants loaded with IFN-( directly in T47D and MCF7 cell lines have not been autologous mesenchymal stem cells on the healing of canine identified yet. Breast cancer cells treated directly by TNF-" and segmental bone defects. J. Bone Joint Surg., 80: 985-996. 9. Kim, N. and S.G. Cho, 2013. Clinical applications of IFN-( showed that these cytokines inhibit the cell growth due mesenchymal stem cells. Korean J. Int. Med., 28: 387-402. to apoptotic or necrotic cells. The hWJMSCs treated by TNF-" 10. Loebinger, M.R., A. Eddaoudi and D. Davies and S.M. Janes, and IFN- showed these cytokines non-toxic against normal ( 2009. Mesenchymal stem cell delivery of TRAIL can eliminate cells. The apoptotic mechanism by direct TNF-" and IFN-( metastatic cancer. Cancer Res., 69: 4134-4142. treatment in T47D and MCF7 should be continued and 11. Grisendi, G., R. Bussolari, L. Cafarelli, I. Petak and V. Rasini et al., clarified especially by the apoptotic analysis 2010. Adipose-derived mesenchymal stem cells as stable and indirectly effect of TNF-" and IFN-( in T47D and MCF7 by source of -related apoptosis-inducing involving Natural Killer (NK) cell as immunemodulatory. ligand delivery for cancer therapy. Cancer Res., 70: 3718-3729. 12. Gjorgieva, D., N. Zaidman and D. Bosnakovski, 2013. Mesenchymal stem cells for anti-cancer drug delivery. Recent ACKNOWLEDGMENTS Patents Anti-Cancer Drug Discovery, 8: 310-318. 13. Ding, D.C., Y.H. Chang, W.C. Shyu and S.Z. Lin, 2015. Human We gratefully acknowledge the financial support of Hibah umbilical cord mesenchymal stem cells: A new era for stem Kompetensi 2015 from Directorate of Higher Education cell therapy. Cell Transplant, 24: 339-347. Indonesia (DIPA DIKTI No. DIPA-023-04.1.673453/2015). This 14. Bongso, A. and C.Y. Fong, 2013. The therapeutic potential, study was supported by Biomolecular and Biomedical challenges and future clinical directions of stem cells from the wharton's jelly of the human umbilical cord. Stem Cell Rev., Research Center, Aretha Medika Utama, Bandung, West Java, 9: 226-240. Indonesia and Stem Cells and Cancer Institute, Jakarta 15. Widowati, W., L. Wijaya, H. Murti, H. Widyastuti and Indonesia for laboratory facilities. We acknowledge to Pande D. Agustina et al., 2015. Conditioned medium from Putu Erawijantari from Biomolecular and Biomedical Research normoxia (WJMSCs-norCM) and hypoxia-treated WJMSCs Center, Aretha Medika Utama, Bandung, West Java, Indonesia (WJMSCs-hypoCM) in inhibiting cancer cell proliferation. for data analysis. Biomarkers Genomic Med., 7: 8-17.

9 Asian J. Cell Biol., 11 (1): 1-12, 2016

16. Shah, K., 2012. Mesenchymal stem cells engineered for cancer 28. Micheau, O. and J. Tschopp, 2003. Induction of TNF receptor therapy. Adv. Drug Deliv. Rev., 64: 739-748. I-mediated apoptosis via two sequential signaling complexes. 17. Menon, L.G., K. Kelly, H.W. Yang, S.K. Kim, P.M. Black and Cell, 114: 181-190. R.S. Carroll, 2009. Human bone marrow-derived 29. Schneider-Brachert, W., V. Tchikov, J. Neumeyer, M. Jakob and mesenchymal stromal cells expressing S-TRAIL as a cellular S. Winoto-Morbach et al., 2004. Compartmentalization of TNF delivery vehicle for human glioma therapy. Stem Cells, receptor 1 signaling: Internalized TNF receptosomes as death 27: 2320-2330. signaling vesicles. Immunity, 21: 415-428. 18. Curnis, F., A. Sacchi, L. Borgna, F. Magni, A. Gasparri and 30. Schutze, S., V. Tchikov and W. Schneider-Brachert, 2008. A. Corti, 2000. Enhancement of tumor necrosis factor " Regulation of TNFR1 and CD95 signalling by receptor antitumor immunotherapeutic properties by targeted compartmentalization. Nat. Rev. Mol. Cell Biol., 9: 655-662. delivery to aminopeptidase N (CD13). Nat. Biotechnol., 31. Marques-Fernandez, F., L. Planells-Ferrer, R. Gozzelino, 18: 1185-1190. K.M.O. Galenkamp and S. Rei et al., 2013. TNF" induces 19. Lupertz, R., Y. Chovolou, A. Kampkotter, W. Watjen and survival through the FLIP-L-dependent activation of the R. Kahl, 2008. Catalase overexpression impairs TNF-" induced MAPK/ERK pathway. Cell Death Dis., 10.1038/cddis.2013.25. NF-6B activation and sensitizes MCCF-7 cells against TNF-". 32. Wong, V.K.W., M.M. Zhang, H. Zhou, K.Y.C.L and P.L. Chan, J. Cell Biochem., 103: 1497-1511. 2013. Saikosaponin-d enhances the anticancer potency of 20. Battegay, E.J., E.W. Raines, T. Colbert and R. Ross, 1995. TNF-" via overcoming Its undesirable response of activating TNF-alpha stimulation of fibroblast proliferation. Dependence NF-5 B signalling in cancer cells. Evid. Based Complement on platelet-derived growth factor (PDGF) secretion and Altern. Med., 10.1155/2013/745295 alteration of PDGF receptor expression. J. Immunol., 33. Pileczki, V., C. Braicu, C.D. Gherman and I. Berindan-Neagoe, 154: 6040-6047. 2013. TNF-" in triple negative breast cancer 21. Teixeira, L.K., B.P. Fonseca, A. Vieira-de-Abreu, B.A. Barboza, cell line induces apoptosis. Int. J. Mol. Sci., 14: 411-420. B.K. Robbs, P.T. Bozza and J.P. Viola, 2005. IFN-gamma 34. Roberts, N.J., S. Zhou, L.A. Diaz Jr. and M. Holdhoff, 2011. production by CD8+ T cells depends on NFAT1 transcription Systemic use of tumor necrosis factor alpha as an anticancer factor and regulates Th differentiation. J. Immunol., agent. Oncotarget, 2: 739-751. 175: 5931-5939. 35. Sasi, S.P., X. Yan, H. Enderling, D. Park and H.Y. Gilbert et al., 22. Schmitt, M.J., D. Philippidou, S.E. Reinsbach, C. Margue and 2012. Breaking the 'harmony' of TNF-" signaling for cancer A. Wienecke-Baldacchino et al., 2012. Interferon-(-induced treatment. , 31: 4117-4127. activation of Signal Transducer and Activator of Transcription 36. Gupta, S., 2002. A decision between life and death during 1 (STAT1) up-regulates the tumor suppressing microRNA-29 TNF-"-induced signaling. J. Clin. Immunol., 22: 185-194. family in melanoma cells. Cell Commun. Signal, 37. Canbay, A., S. Friedman and G.J. Gores,, 2004. Apoptosis: 10.1186/1478-811X-10-41. The nexus of liver injury and fibrosis. Hepatology, 39: 273-278. 23. Widowati, W., L. Wijaya, I. Bachtiar, R.F. Gunanegara and 38. Wajant, H., K. Pfizenmaier and P. Scheurich, 2003. Tumor S.U. Sugeng et al., 2014. Effect of oxygen tension on necrosis factor signaling. Cell Death Differ., 10: 45-65. proliferation and characteristics of Wharton's jelly-derived 39. Walter, D., K. Schmich, S. Vogel, R. Pick and T. Kaufmann et al., mesenchymal stem cells. Biomarkers Genomic Med., 6: 43-48. 2008. Switch from type II to I Fas/CD95 death signaling 24. Widowati, W., T. Mozef, C. Risdian and Y. Yellianty, 2013. on in vitro culturing of primary hepatocytes. Hepatology, Anticancer and free radical scavenging potency of 48: 1942-1953. Catharanthus roseus, Dendrophthoe petandra, Piper betle 40. Ghavami, S., M. Eshraghi, K. Kadkhoda, M.M. Mutawe and and Curcuma mangga extracts in breast cancer cell lines. S. Maddika et al., 2009. Role of BNIP3 in TNF-induced cell Oxidants Antioxid. Med. Sci., 2: 137-142. death-TNF upregulates BNIP3 expression. Biochimica 25. Widowati, W., L. Wijaya, T.L. Wargasetia, I. Bachtiar, Y. Yellianty Biophysica Acta (BBA)-Mol. Cell Res., 1793: 546-560. and D.R. Laksmitawati, 2013. Antioxidant, anticancer and 41. Huang, J., L. Wu, S.I. Tashiro, S. Onodera and T. Ikejima, 2005. apoptosis-inducing effects of Piper extracts in HeLa cells. The augmentation of TNF"-induced cell death in murine J. Exp. Integr. Med., 3: 225-230. L929 fibrosarcoma by pan-caspase inhibitor Z-VAD-fmk 26. Shen, H.M. and S. Pervaiz, 2006. TNF receptor through premiochondrial and MAPK-dependent pathways. superfamily-induced cell death: Redox-dependent execution. Acta Med. Okayama, 59: 253-260. FASEB J., 20: 1589-1598. 42. Boldin, M., T. Goncharov, Y.V. Goltsev and D. Wallach, 1996. 27. Van Horssen, R., T.L.M. ten Hagen and A.M.M. Eggermont, Involvement of MACH, a Novel MORT1/FADD-Interacting 2006. TNF-" in cancer treatment: Molecular insights, Protease, in Fas/APO-1- and TNF receptor-induced cell death. antitumor effects and clinical utility. Oncologist, 11: 397-408. Cell, 85: 803-815.

10 Asian J. Cell Biol., 11 (1): 1-12, 2016

43. Varfolomeev, E.E., M. Schuchmann, V. Luria, N. Chiannilkulchai 57. Kim, K.S., K.J. Choi and S. Bae, 2012. Interferon-( enhances and J.S. Beckmann et al., 1998. Targeted disruption of the radiation-induced cell death via downregulation of Chk1. mouse Caspase 8 gene ablates cell death induction by the Cancer Biol. Ther., 13: 1018-1025. TNF receptors, Fas/Apo1 and DR3 and is lethal prenatally. 58. Chawla-Sarkar, M., D.J. Lindner, Y.F. Liu, B.R. Williams, G.C. Sen, Immunity, 9: 267-276. R.H. Silverman and E.C. Borden, 2003. Apoptosis and 44. Faleiro, L., R. Kobayashi, H. Fearnhead and Y. Lazebniket, 1997. interferons: Role of interferon-stimulated genes as mediators Multiple species of CPP32 and Mch2 are the major active of apoptosis. Apoptosis, 8: 237-249. caspases present in apoptotic cells. EMBO J., 16: 2271-2281. 59. Ikeda, H., L.J. Old and R.D. Schreiber, 2002. The roles of IFN( in 45. Cryns, V. And J. Yuan, 1998. Proteases to die for. Genes Dev., protection against tumor development and cancer 12: 1551-1570. immunoediting. Cytokine Growth Factor Rev., 13: 95-109. 46. Lin, Y., S. Choksi, H.M. Shen, Q.F. Yang and G.M. Hur et al., 60. Chin, Y.E., M. Kitagawa, W.C. Su, Z.H. You, Y. Iwamoto and 2004. Tumor necrosis factor-induced nonapoptotic cell death X.Y. Fu, 1996. Cell growth arrest and induction of requires receptor-interacting protein-mediated cellular cyclin-dependent kinase inhibitor p21WAF1/CIP1 mediated reactive oxygen species accumulation. J. Biol. Chem., by STAT1. Science, 272: 719-722. 279: 10822-10828. 61. Hobeika, A.C., W. Etienne, B.A. Torres, H. M. Johnson and 47. Li, H., H. Zhu, C.L. Xu and J. Yuan, 1998. Cleavage of BID by P.S. Subramaniam, 1999. IFN-( induction of p21WAF1 is caspase 8 mediates the mitochondrial damage in the Fas required for cell cycle inhibition and suppression of pathway of apoptosis. Cell, 94: 491-501. apoptosis. J. Interferon Cytokine Res., 12: 1351-1361. 48. Luo, X., I. Budihardjo, H. Zou, C. Slaughter and X. Wang, 1998. 62. Lee, S.H., J.W. Kim, H.W. Lee, Y.S. Cho and S.H. Oh et al., 2003. Bid, a Bcl2 interacting protein, mediates cytochrome c release Interferon regulatory factor-1 (IRF-1) is a mediator for from mitochondria in response to activation of cell surface interferon-( induced attenuation of telomerase activity and death receptors. Cell, 94: 481-490. human telomerase reverse transcriptase (hTERT) expression. 49. Wang, L., F. Du and X. Wang, 2008. TNF-" induces two distinct Oncogen, 22: 381-391. caspase-8 activation pathways. Cell, 133: 693-703. 63. Kortylewski, M., W. Komyod, M.E. Kauffmann, A. Bosserhoff, 50. Hehlgans, T. and K. Pfeffer, 2005. The intriguing biology of the P.C. Heinrich and I. Behrmann, 2004. Interferon-(-mediated tumour necrosis factor/tumour necrosis factor receptor growth regulation of melanoma cells: involvement of superfamily: Players, rules and the games. Immunology, STAT1-dependent and STAT1-independent signals. 115: 1-20. J. Invest. Dermatol., 122: 414-422. 51. Totzke, G., K. Schulze-Osthoff and R.U. Janicke, 2003. 64. Kayagaki, N., N. Yamaguchi, M. Nakayama, H. Eto, K. Okumura Cyclooxygenase-2 (COX-2) inhibitors sensitize tumor cells and H. Yagita, 1999. Type I interferons (IFNs) regulate tumor specifically to death receptor-induced apoptosis necrosis factor-related apoptosis-inducing ligand (TRAIL) independently of COX-2 inhibition. Oncogene, 22: 8021-8030. expression on human T cells: A novel mechanism for the 52. Machuca, C., C. Mendoza-Milla, E. Cordova, S. Mejia, antitumor effects of type I IFNs. J. Exp. Med., 189: 1451-1460. L. Covarrubias, J. Ventura and A. Zentella, 2006. 65. Chen, Q., B. Gong, S. Ashraf. M. Ahmed, A. Zhou and Dexamethasone protection from TNF-alpha-induced cell E.D. His et al., 2001. Apo2L/TRAIL and Bcl-2-related proteins death in MCF-7 cells requires NF-kappaB and is independent regulate type I interferon-induced apoptosis in multiple from AKT. BMC Cell Boil., 10.1186/1471-2121-7-9 53. Aggarwal, B.B., T.E. Eessalu and P.E. Hass, 1985. myeloma. Blood., 98: 2183-2192. Characterization of receptors for human tumour necrosis 66. Balachandran, S., P.C. Roberts, T. Kipperman, K.N. Bhalla, R.W. Compans, D.R. Archer and G.N. Barber, 2000. Alpha/beta factor and their regulation by (-interferon. Nature, 318: 665-667. interferons potentiate virus-induced apoptosis through 54. Matsubara, N., S. Fuchimoto and K. Orita, 1991. activation of the FADD/Caspase-8 death signaling pathway. Antiproliferative effects of natural human tumor necrosis J. Virol., 74: 1513-1523. factor-", interferon-" and interferon-( on human pancreatic 67. Tsuno, T., J. Mejido, T. Zhao, H. Schmeisser, A. Morrow and carcinoma cell lines. Int. J. Pancreatol., 8: 235-243. K.C. Zoon, 2009. IRF9 is a key factor for eliciting the 55. Dunn, G.P., C.M. Koebel and R.D. Schreiber, 2006. Interferons, antiproliferative activity of IFN-". J Immunother., immunity and cancer immunoediting. Nat. Rev. Immunol., 32: 803-816. 6: 836-848. 68. Morrison, B.H., J.A. Bauer, D.V. Kalvakolanu and D.J. Lindner, 56. Schreiber, R.D., L.J. Old and M.J. Smyth, 2011. Cancer 2001. Inositol hexakisphosphate kinase 2 mediates growth immunoediting: Integrating immunity's roles in cancer suppressive and apoptotic effects of interferon-$ in ovarian suppression and promotion. Science, 331: 1565-1570. carcinoma cells. J. Biol. Chem., 276: 24965-24970.

11 Asian J. Cell Biol., 11 (1): 1-12, 2016

69. Tekautz, T.M., K. Zhu, J. Grenet, D. Kaushal, V.J. Kidd and 73. English, K., F.P. Barry, C.P. Field-Corbett and B.P. Mahon, 2007. J.M. Lahti, 2006. Evaluation of IFN-( effects on apoptosis and IFN-( and TNF-" differentially regulate immunomodulation gene expression in -Preclinical studies. by murine mesenchymal stem cells. Immunol. Lett., Biochimica et Biophysica Acta (BBA)-Mol. Cell Res., 110: 91-100. 1763: 1000-1010. 74. Hill, J.J., T.L. Tremblay, C. Cantin, M. O'Connor-McCourt, 70. Schroder, K., P.J. Hertzog, T. Ravasi and D.A. Hume, 2004. J.F. Kelly and A.E. Lenferink, 2009. Glycoproteomic analysis of Interferon-(: An overview of signals, mechanisms and two mouse mammary cell lines during transforming growth functions. J. Leukoc. Biol., 75: 163-189. factor (TGF)-$ induced epithelial to mesenchymal transition. 71. Prasanna, S.J., D. Gopalakrishnan, S.R. Shankar and Proteome Sci., Vol. 7. A.B. Vasandan, 2010. Pro-inflammatory cytokines, IFN( and 75. Duijvestein, M., M.E. Wildenberg, M.M. Welling, S. Hennink TNF", influence immune properties of human bone marrow and I. Molendijk et al., 2011. Pretreatment with Interferon ( and Wharton jelly mesenchymal stem cells differentially. enhances the therapeutic activity of mesenchymal stromal PLoS One, 10.1371/journal.pone.0009016. cells in animal models of colitis. Stem Cells, 29: 1549-1558. 72. Kim, K.S., K.W. Kang, Y.B. Seu, S.H. Baek and J.R. Kim, 2009. Interferon-( induces cellular senescence through p53-dependent DNA damage signaling in human endothelial cells. Mech. Ageing Dev., 130: 179-188.

12